Agents targeting topoisomerase II are active against a wide range of human tumors. Stabilization of covalent complexes, converting topoisomerase II into DNA damage, is an essential aspect of cell killing by these drugs. We have taken advantage of newly developed yeast genomic tools to identify genes encoding DNA repair or DNA damage tolerance functions that play key roles in sensitivity to drugs targeting topoisomerases. Successful utilization of these tools, notably a set of strains deleting all non-essential open reading frames has required us to develop new approaches to efficiently enhance the sensitivity of yeast cells to drugs targeting topoisomerase II. We developed several strategies for dominantly increasing the sensitivity of yeast cells to topoisomerase targeting drugs, including the development of chimeric transcriptional regulators that repress the expression of drug efflux genes. Recent findings that have taken advantage of the yeast deletion set have demonstrated that the nuclease activities of several repair complexes are important following cell survival following exposure to Top2 targeting agents. We have also shown that proteins that regulate protein stability following exposure to DNA damage also play key roles in repairing Top2 mediated DNA damage. During the next grant period we will use these tools to identify yeast genes that affect cell survival following exposure to Top2 targeting drugs, but do not affect sensitivity to other types of DNA damage. Experiments will elucidate the biochemical roles of proteins that process the DNA damage generated by topoisomerases. These proteins include factors that regulate the stability of RNA polymerase in response to DNA damage and nucleases that are specific for altered DNA structures. These studies of the role of DNA repair functions on sensitivity to topoisomerase targeting agents have the long-term goal of understanding factors that contribute to the efficacy of clinically important agents such as etoposide and doxorubicin. The results from experiments in this proposal should enhance our understanding of the mechanisms of action of these drugs targeting DNA topoisomerases. A key question that this work will address is why drugs targeting different topoisomerases have different effects on cell survival, and therefore different clinical efficacies. Answering these questions may also suggest strategies for circumventing resistance to these clinically important anticancer drugs.